Despite significant advances in the surgical and medical management of congenital heart disease, congenital cardiac anomalies remain a leading cause of death in the newborn period. Most severe forms of congenital heart disease require surgical intervention. Complications arising from the use of currently available prosthetic materials in the form of vascular grafts, patches, or replacement heart valves are a leading source of morbidity and mortality after congenital heart surgery. Currently available prosthetic materials such as polytetraflouroethylene (PTFE) are a significant source of thromboembolism, have poor durability due to neointimal hyperplasia, are susceptible to infection, and perhaps most importantly lack growth capacity, which results in the need for additional operations as children outgrow their prosthetics. The development of better biomaterials with growth potential could substantially improve the outcomes of children requiring congenital heart surgery by reducing the number of graft-related complications and enabling earlier definitive surgical repair without the risk of serial re-operation. Tissue engineering offers an innovative solution to this significant problem. Using tissue engineering methods, bioprosthetics can be made from an individual's own cells creating a living material with excellent biocompatibility and the ability t grow, repair, and remodel. We previously created the first tissue engineered vascular graft (TEVG) specifically designed for use in children, where its growth potential could be used to its greatest advantage. We subsequently performed the first clinical trial evaluating the use of the TEVG in congenital heart surgery. In this study we confirmed the growth capacity of the TEVG but also demonstrated that stenosis was the primary graft-related complication and the critical barrier preventing the widespread clinical adaptation of this technology. Our goal is to optimize the design of our TEVG. Using the TEVG as a model for bioprosthetics specifically developed for use in congenital heart surgery, we will investigate the fundamental mechanisms that lead to stenosis, the principal limitation of current TEVGs, and then apply our discoveries to the design of an improved vascular graft. In this application, we will focus on the role of transforming growth factor-? (TGF-?), which we have previously demonstrated is critical for the formation of TEVG stenosis in mouse models. We will explore the role of this cytokine both upon infiltrating host macrophages (aim 1) and then upon vascular cells that form the stenotic lesions (aim 2), applying the power of mouse genetics to dissect these processes. Finally, because findings in mice do not always effectively translate into the clinic, we will validate our findings in both a FDA-required ovine model and in a humanized mouse model (aim 3). Successful completion of this work will facilitate the further development and translation of this promising technology from the bench to the bedside for use in the repair of congenital cardiac anomalies and overcome a critical barrier preventing its widespread use. It would also open the door and provide a pathway for creating more complex tissue-engineered cardiovascular structures such as valved- conduits, which could further improve our abilities to treat and even cure the most severe forms of congenital heart disease.